The goal of this project is to create a new class of potent toxins that selectively target HIV and HIV-infected cells for destruction. The toxin, an engineered bacterial ribonuclease, is catalytically inactive in uninfected cells and does not harm them. The ribonuclease is activated upon cleavage by HIV protease. The method for suppressing and activating catalytic activity is based on a novel forced-unfolding mechanism. Once activated, the enzyme disrupts HIV infection by three independent mechanisms. First, it kills infected cells by degrading cytosolic RNA. Second, it is packaged into the HIV capsid, where it destroys the HIV RNA genome. Third, it is activated and delivered to cells by the HIV particle itself, upon subsequent rounds of infection. These effects are cumulative. The net result is that HIV infection is halted after a single round. By attacking the virus as well as the cells that harbor it, the proposed method is designed to rid HIV completely from the body, especially when used in conjunction with therapies that activate HIV gene expression in latently-infected cells. The first aim describes the design, construction and in vitro testing of two separate designs of the toxin. The proteins are optimized so that ribonuclease activity is high in the protease-cleaved state and undetectable in the uncleaved form. In the second aim, each of the three anti-HIV mechanisms is tested ex vivo. Toxins are delivered into cultured human cells by direct transduction (facilitated by fusion to the cell-penetrating HIV TAT peptide) as well as by a viral gene vector. The cells are then infected with HIV. Cell viability and viral infectivity assays determine the extent to which the toxins selectively kill infected cells and inactivate HIV. A major benefit of this therapy is that the molecules developed here will retain their potency against viral mutation to a much greater degree than existing protease and reverse transcriptase inhibitors. This study creates a new class of molecules that specifically kill HIV and HIV-infected cells, and do not harm healthy cells. These molecules are engineered to remain potent despite viral mutation. This therapy is designed to eliminate the virus as well as diseased cells from the body. [unreadable] [unreadable] [unreadable] [unreadable]